Anti-Cancer Effect of Bromelain and Its Combination with Cisplatin on HN5 Cell Line (Squamous Cell Carcinoma)

Statement of the Problem: Squamous cell carcinoma (SCC) comprises over 90% of oral malignancies. Cisplatin, as a selective chemotherapy agent to treat SCC, has many side effects despite its high effectiveness. There are some studies on the effects of bromelain derived from pineapple stems on different malignancies. Purpose: The aim of this study was to investigate the effect of bromelain alone and in combination with Cisplatin on oral squamous cell carcinoma (OSCC) and fibroblast cell lines. Materials and Method: In this interventional study, the HN5 cell line of OSCC and fibroblast cell line were treated with different concentrations of bromelain alone and in combination with cisplatin. Cell viability test was performed after 24, 48 and 72 hours using MTT (3-)4,5-dimethylthiazol-2-yl(-2,5 diphenyl tetrazolium bromide) assay. In the final stage, the drug-treated cells underwent flow cytometry to assess apoptosis patterns. Data were analyzed using SPSS 17, ANOVA (for general comparison of groups) and LSD post hoc tests (for comparison two groups). p< 0.05 was considered statistically significant. Results: The findings suggested that although bromelain showed toxic effects on HN5 cancer cells, its combination with Cisplatin resulted in little improvement in its effectiveness. Bromelain alone and in combination with Cisplatin presented cytotoxic effects against fibroblasts, which depended on the dosage and time exposure (p< 0.05). The flow cytometry results did not support the superior effect of the combination of two medications over Cisplatin alone (p> 0.05). Conclusion: According to the findings, although adding bromelain to Cisplatin reduced toxicity on normal tissues, the combination of these two drugs did not increase the anticancer effect of Cisplatin. Thus, bromelain in combination with Cisplatin is not recommended as an adjuvant drug for OSCC.

from the growth of malignant cells of the stratified squamous epithelium of the oral mucosa [2]. According to the Center for Disease Control of the Ministry of Health (Iran), SCC is among the 13th most common cancers in Iran [3]. The risk of being diagnosed with oral and pharynx cancer increases by aging, especially in men [2]. Though the diagnostic techniques have progressed, SCC has a high incidence in different parts of the world. Recent studies have represented an increased incidence rate of oral squamous cell carcinoma (OSCC) in Iranian young patients [3]. Oral carcinomas are usually treated with surgery in the primary stages. The intermediate to advanced tumors are usually cured using surgery, radiotherapy, or chemo-radiotherapy. When the disease is highly advanced or surgical process fails to deliver acceptable results, patients may be treated by radiotherapy or chemo-radiotherapy modalities [2].
Cisplatin (Cis-[Pt (11) (NH(3))(2)C1(2)] [PtC12 (NH3) or 2 CDDP)) is one of the most effective chemotherapy treatments that is widely used to treat cancer [4]. Cisplatin (cis-diamminedichloridoplatinum (II) or (CDDP)) is a metallic composition, initially synthesized through M.Peyrone in 1844 and its chemical structure has been firstly discovered by Alfred Werner in 1893 [5]. Cisplatin as a first-generation anti-cancer combination plays a key role in treatment of different types of cancer; moreover, it is a determining treatment factor in head and neck cancers, especially OSCC, which has been widely used to treat this disease [6][7][8]. A significant number of patients diagnosed with different types of cancers such as sarcomas, cancers of soft tissue, bones, muscles, and blood vessels have been successfully cured by the use of Cisplatin [9]. Its mechanism is related to the DNA and transcription inhibition [10].
However, the usage of Cisplatin is limited due to the side effects on normal tissues [11].
Pineapple is the common term for Ananas Comosus, which is an edible member of Bromeliaceae family and has been used as a medical herb in several indigenous cultures [12]. The crude aqueous extract from stem and fruit of pineapple is known as bromelain. This extract is comprised of different thiol endopeptidases and other components including glucosidases, phosphatases, peroxidases, carbohydrates, celluloses, glycoproteins, and several protease regulators [13]. Recent studies suggest that bromelain has the ability to modify the key path-ways of creating cancer. The anti-cancer activity of bromelain is the direct impact on cancer cells and their microenvironment, as well as the modulation of immune, inflammatory, and hemostatic systems. Probably, the anticancer activity of bromelain is due to its direct effect on cancer cells and their microenvironment, as well as the modulation of immune, inflammatory, and hemostatic systems [14]. In an experiment by Taussig et al. [15], mouse skin papilloma was treated with bromelain; it was found that bromelain could reduce tumor formation, tumor volume and cause apoptotic cell death.
In one study [16] performed on treatment with bromelain in gastric carcinoma cell lines, KATO III showed a significant reduction in cell growth, while in another study [17], bromelain reduced the potential for glioblastoma cell invasion and reduced de novo protein synthesis. Bromelain overexpresses the p53 and Bax, which are known as apoptotic activators in the skin of mice [18]. Bromelain also reduces the activity of cell survival regulators such as Akt and Erk, thus increasing apoptosis in tumors. Various studies have shown the role of NF-κB, Cox-2, and PGE2 as promoters of cancer progression. Evidence suggests that NF-κB signaling and overexpression play an important role in many types of cancer [19][20]. Cox-2, a multifunctional gene from NF-κB, facilitates the conversion of arachidonic acid to PGE2 and thus enhances angiogenesis and tumor progression [18]. Inhibition of NF-κB, Cox-2, and PGE2 activity is thought to be a potential cancer treatment.
Bromelain has been shown to downregulate NF-κB and Cox-2 in rat papilloma and skin tumorigenic models [18]. Bromelain has also been shown to inhibit NF-κB activity induced by bacterial endotoxin (LPS) as well as the expression of PGE2 and Cox-2 in human monocytic leukemia and murine microglial cell lines [21][22].
Chang et al. [23], showed that bromelain, as part of its antiproliferative mechanisms, increased oxidative stress and superoxide production by 6-fold in bromelain-treated human colon cancer cells. A recent study [23], also reported the effect of in vitro bromelain on macrophage pathway activation and lysosome formation via increasing autophagy-associated proteins levels (ATG5 / 12, beclin, p62, and LC3I / II) leading to apoptosis. The microenvironmental anti-inflammatory properties of bromelain by uncoated cancer cells (depolymerizing MUC-1, fibrin and albumin) through in-creasing adhesion of lymphocytes to the tumor are thought to expose the tumor to host defense [24]. Bromelain can neutralize overexpression of the oncoglycoprotein MUC-1, which promotes the proliferation and enhancement of antiapoptotic properties of cancer cells along with invasion and chemical resistance in various human tumors. In addition, bromelain has been shown to cause radiation sensitivity in the 4T1 mouse breastcancer cell line [24].
Many vaccine and immunotherapy development programs aim to modulate immune responses. Bromelain is a mixture of cysteine proteases that modulate immune responses [25]. In a study by Engwerda et al. This provides important insights into the immunemodulating activity of bromelain [25].
In another study by Engwerda et al. [26], bromelain was shown to increase the production of IFN-γmediated nitric oxide and TNFα by macrophages. Bromelain can also increase IL-2 and IL-12-mediated IFN-γ production by NK cells [26]. These results indicate the potential role of bromelain in activating inflammatory responses in situations where individuals may have immunodeficiency [26].
Fouz et al. [27] investigated the autophagy phenomenon in carcinoma cells of breast under treatment by bromelain and the relationship between autophagy and apoptosis in MCF-7 cells. MCF-7 cells exposed to bromelain represented delayed growth inhibitory response and inductive apoptosis, which was detected using monodansylcadaverine. Apoptotic cell death was clearly discovered [26]. Pillai et al. [28]  The results have illustrated that bromelain effectively reduces cancer cells proliferation via apoptosis [29].
Mekkawy et al. [30] presumed that bromelain could act as a radiosensitizer for tumor cells. To prove this hypothesis, MTT cell proliferation in vitro was used and showed that pretreatment with bromelain could sensitize irradiated Ehrlich ascites carcinoma (EAC) cells. In their study, animals were divided into five groups and treated with different doses of radiation and bromelain [30]. Histopathological examination of mice with Ehrlich solid tumor (EST) that underwent gamma radiation and bromelain treatment showed significantly reduced size and weight of the tumors. Therefore, they concluded that bromelain could be considered a radiosensitizer and radioprotector that plays an important role in reducing the radiation dose during radiation therapy [30]. In addition, Chermahini et al. [31], examined bromelain, The acquired data were analyzed using SPSS v17, ANOVA tests (for general comparison of groups) and post-hoc LSD tests (for comparison of two groups), and p< 0.05 was considered statistically significant [35].
On the microscopic study, a cell deformation was observed so that the cell viability status could be considered ( Figure 3). Flow cytometry experiments were conducted to evaluate the effects of different concentrations of medicines on HN5 cell lines and fibroblast without repetition. In flow cytometry, no significant difference between sub-groups of necrosis, apoptosis and live cells were found after 48 hours (Figure 4).

Discussion
The results showed that bromelain had toxic effects on HN5 cancerous cells so that its combination with Cisplatin did not much improve its effectiveness. Nowadays, the usage of herbal products as a supplementary medicine for curing various illnesses is increasing due to the numerous side effects of chemical medicines [18].
Meanwhile, considering the prevalence of OSCC and numerous side effects of chemical medicines, the use of herbal medicines or in combination with effective classic medicines may lead to less side effects during the treatment and overcome medical resistance [16,18].
Bromelain is a protease enzymes derived from pineapp- le essence that has anti-cancer effects such as anticarcinoma in various studies [16,18]. The aim of the present study was to find a proper dosage of bromelain in combination with Cisplatin, which has a toxic effect on cancerous cells and decreases its related side effects on normal cells.
The results showed that bromelain with different concentrations could destroy cancerous cells, as previous studies already represented [28,31,35]. Nevertheless, this effect entailed cytotoxicity against normal fibroblasts, which was enhanced in cancer cells and increased with time and increasing doses in both cancer cells and fibroblasts. It was found that bromelain in high concentrations had toxic effects on HN5 cell lines, which is consistent with the results of Amini et al. [16] who found the cytotoxic effects of bromelain on MKN-45, KATO-III cell lines of gastric carcinoma and HT29-5M21, HT29-5F12 cell lines of adenocarcinoma colon. However, they did not investigate the effects of the drugs on normal cells. The present study has suggested that the concentrations of drugs, which can reduce viability of cancer cells to half the initial value, have cytotoxicity effect on fibroblasts even more on cancer cells.
Like the current study, Manosroi et al. [36] stated that the cytotoxicity effect of bromelain on cancerous cells was less than that of Cisplatin.
Besides, bromelain in combination with Cisplatin had no effect on HN5 cell line. Unlike the present results, the findings of Pauzi et al. [35], who investigated the toxicity of the combination of bromelain and Cisplatin, displayed synergistic effects against MDA-MB-231 cells.
Additionally, Pillai et al. [28] revealed that adding bromelain significantly increased the toxicity of potassium Cisplatin in malignant peritoneal mesothelioma cells.
Chermahini et al. [31]  hours after treatment of the cells. Overall, there were no significant differences between sub-groups of apoptosis, necrosis, and live cells [35].
The main difference between the current study and other similar ones [27,37] was the effect of using synchronous medicines on cancerous cell lines and normal fibroblast. Although some of the processes occurring in the cancer progression such as cell proliferation and apoptosis are dependent on proteases, the proteolytic activity in tumors is regulated in a complex manner, because the cancer and stroma cells including fibroblasts, endothelial and inflammatory cells are involved [38]. However, clinical trials using protease inhibitors have far been unsuccessful except for a few applications of matrix metalloprotease (MMP) inhibitors when used in combination with cytostatic anticancer agents and/or in the early stages of cancer [38].
Since the proteolytic effects of bromelain have been reported in vitro and no strong evidence is available for in vivo effects , and concerning that most in vitro studies have been performed without considering normal cells (such as normal fibroblasts), it seems that bromelaininduced cytotoxicity has a direct proteolytic effect and is not a unique mechanism to counter cancer cells. This confirms that bromelain causes more cytotoxicity on normal cells than cancerous ones.
At the end, it can be concluded that even though bro-